Techniques applicable to the generation of complementary DNA (cDNA) to specific messenger RNA (mRNA) provide an important mechanism by which the structure, organization and expression of genetic material in eukaryotic cells can be analyzed. More particularly, the mechanism enables the identification isolation and characterization of specific genes resident in chromosomal DNA. One generalized method by which cDNA can be synthesized is the contacting of a heterogenous mixture of mRNA to reverse transcriptase to generate cDNA:mRNA hybrid molecules. The RNA strands are removed from the hybrids by heat denaturation or by RNase H treatment and the second cDNA strand is synthesized using DNA polymerase (Klenow fragment) and S1 nuclease treatment. The resulting hetergeneous mixture of double stranded cDNA molecules can then be cloned using a variety of techniques into recombinant DNA vector molecules.
In an alternative approach, mRNA molecules are first annealed to vector DNA. Okayama and Berg I (Mol. Cell. Biol 2: 161-170, 1982), and incorporated herein by reference, have described such a strategy for cloning full length, or nearly full length, cDNA molecules in Escherichia coli. This cloning strategy is predicated on linking an oligo (dT) primer to a linearized vector to generate a vector primer and the annealing poly (A).sup.+ mRNA to the oligo (dT) primer. A cDNA strand is synthesized complementary to the annealed mRNA template and the strand terminated with an oligo (dC) linker. The resulting linear molecule is cleaved with the restriction endonuclease Hind III to yield a molecule unable to circularize either by annealing or by ligation due to the presence of incompatible ends. Consequently, circularization is accomplished by the intermolecular ligation of a linker DNA segment comprising a Hind III generated terminus and a oligo (dG) tailed terminus which allows the abridgement of the vector primer Hind III terminus with the oligo (dC) terminated cDNA:mRNA. In this procedure, the RNA strand is replaced by DNA following treatment with RNase H, DNA polymerase and DNA ligase. Okayama and Berg II (Mol. Cell. Biol. 3: 280-289, 1983), and incorporated herein by reference, subsequently described a cloning vector system based on the aforementioned cloning strategy that permits expression of cDNA in mammalian cells. This vector system comprises pcDV1, a recombinant molecule containing a DNA sequence from pBR322 and two segments from Simian Virus 40 (SV40) including the poly A region. Using this system, pcDV1 is restricted with the enzyme Kpn 1 and ligated to an oligo (dT) linker to generate the vector primer. mRNA is then annealed and the cDNA synthesized as described above. The oligo (dC) tailed linker segment required for re-circularization and which contains the SV40 origin of replication, is derived from plasmio pLl. The recombinant cDNA molecule is thus capable of replication in both E. coli and SV40-sensitive mammalian cells.
By employing the general principles of the Okayama and Berg procedure, alternative strategies for cDNA cloning have been developed. Nakamura, et al. (Gene, 44:347-351, 1986) developed an expression vector useful in immunological screening of desired recombinant molecules. In this system, expression of cloned cDNA is directed by tandem lac control regions derived from pUC8 and pUC19. Carrington and Morris (Virology 139: 22-32, 1984) cloned a portion of the Carnation Mottle virus germ using DNA fragments derived from pVC8 and pBR322 instead of the pBR322-SV40 hybrid plasmid described by Okayama and Berg. In another procedure, Coleclough and Erlitz (Gene 34:305-314, 1985) described the use of primer-restriction-end (PRE) adapters in a cDNA cloning strategy. In this procedure, cDNA is synthesized using a PRE adapter as a primer oligonucleotide. One end of the cDNA strand is annealed to a vector by oligo (dC) and (dG) pairing. The other end is ligated by the addition of another PRE adapter.
Although the high efficiency of incorporation of full length cDNA obtained using the Okayama and Berg protocol is a substantial advantage, this protocol has not been employed in many studies due to the relatively low efficiency with which transformants are obtained per microgram of starting vector and polyadenylated RNA, relative to the use of bacteriophage vectors and the difficulties associated with screening bacterial colonies, relative to bacteriophage plaques, using hybridization probes. The present invention describes recombinant DNA vector molecules which allow the efficient cloning of full length cDNA using a modification or the method of Okayama and Berg. The vectors described herein are also useful in the enrichment of specific sequences directly from cDNA libraries by hybridization/selection.